Deletion of self-reactive T cells before entry into the thymus medulla

The thymus is important in the differentiation of bone marrow-derived precursor cells into functional T cells; humoral factors, as well as physical interactions with nurse cells, dendritic cells and epithelial cells, are thought to be instrumental in this process. Thymic lymphocytes mature during their migration from the cortical to the medullary region of the thymus, when they undergo phenotypic changes that include the acquisitions of T-cell antigen receptors, hormone receptors and differentiation antigens. Cortical T cells are thus mostly CD4+CD8+, whereas medullary T cells are either CD4+CD8- or CD4-CD8+. During this period T cells are subjected to two types of repertoire selection: all T cells recognizing self-MHC with low affinity may be preferentially amplified (positive selection), and in a second step T cells with high-affinity receptors for self-MHC determinants plus self antigens are eliminated (negative selection). We have described two monoclonal antibodies specific for the V beta 6 gene segment of the alpha/beta heterodimeric T-cell antigen receptor and have shown that most CD4+/V beta 6+ T cell recognize the Mlsa antigenic determinant but not Mlsb; similar results have been reported for V beta 8.1 and Mlsa. In both situations, tolerance to Mlsa correlated in an MHC-dependent fashion with absence of V beta 6 or V beta 8.1 T-cell antigen receptor expressing T cells in the periphery. We show here by immunostaining of thymus cryosections and cytofluorometric analysis that V beta 6-expressing cortical T cells are present at high density in both Mlsa and Mlsb mice, but do not enter the medullary region of Mlsa animals.     

Thymic cortical epithelial cells can present self-antigens in vivo

Antigens present during neonatal life are recognized as self and individuals are tolerant to these antigens. In normal individuals T cells are tolerant to most self proteins but we still know little of the mechanism(s) by which tolerance is established. A requisite part of the current negative selection model of self tolerance is the expression of self proteins complexed with major histocompatibility complex molecules in the thymus. As MHC proteins bind antigens and present them to the receptor on the antigen-specific T cell, then for tolerance to self to occur, it is possible that each self protein must be processed and presented by an MHC molecule. As a result of the development of a unique T-cell hybrid reactive to the self protein murine haemoglobin, we have shown that in normal animals this self protein is continuously processed and potentially presented in an MHC-restricted manner. Here we show that self haemoglobin is being processed and presented by thymic antigen-presenting cells as early as gestational day 14. We also demonstrate that three types of thymic stromal cells, namely macrophages, dendritic cells and cortical epithelial cells, can present the haemoglobin self antigen in vivo. This surprising presentation of a self antigen by thymic cortical epithelial cells implies that they could be involved in T-cell development and negative selection.     

Lower receptor avidity required for thymic clonal deletion than for effector T-cell function.

Clonal deletion in the thymus plays a major part in T-cell tolerance to self antigens. But the mechanism of negative selection, its fine specificity and the threshold of affinity and avidity remains unknown. We have now examined these aspects of negative selection with mice expressing a transgenic T-cell receptor with specificity for lymphocytic choriomeningitis virus (LCMV) glycoprotein in association with the class I H-2Db molecule. These mice were rendered tolerant to LCMV by neonatal infection with mutant LCMVs bearing point mutations in the T-cell epitope recognized by the transgenic T-cell receptor. Variant LCMVs were also tested for their ability to elicit antiviral responses in transgenic mice in vivo and in vitro. Comparison in vivo revealed that a low-avidity receptor interaction, which was unable to induce effector T cells in the periphery, was still sufficient for clonal deletion in the thymus.     

Essential requirement for major histocompatibility complex recognition in T-cell tolerance induction

The induction of T-cell responses involves the recognition of extrinsic antigen in association with antigens of the major histocompatibility complex (MHC), in mice and man, with different T cells recognizing antigen in association with either class I (H-2K/D, HLA-A, B, C) or class II (Ia, HLA-D/DR) MHC antigens. However, the requirement of MHC recognition in the induction of immunological tolerance remains ill defined. With human T helper clones recognizing synthetic peptides of influenza haemagglutinin (HA-1), we have investigated the nature of antigen-induced stimulation, and antigen-induced antigen-specific unresponsiveness, immunological tolerance. Tolerance is not due to cell death, as the cells remain responsive to interleukin-2 and is associated with the loss of T3 antigen from the cell surface. Using monoclonal antibodies to the non-polymorphic regions of human class II antigens to inhibit the induction of T-cell tolerance we report here that induction of tolerance requires the recognition of MHC antigens.     

A cell line that can induce thymocyte positive selection.

The thymus positively selects thymocytes that bear T-cell receptors which recognize antigen presented by self major histocompatibility complex (MHC) proteins. Positive selection is usually driven by MHC products on radiation-resistant cortical epithelial cells. It is unknown whether positive selection is mediated by all thymic epithelial cells or by some specialized subsets. Here we introduce an H-2b-expressing thymic epithelial cell line into the thymuses of lethally irradiated H-2k animals reconstituted with H-2b/k F1 BM or fetal liver cells. I-Ab-restricted T cells are found in these animals, demonstrating that selection occurs on the introduced epithelial cells.     

Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen

The crucial role of the thymus in immunological tolerance has been demonstrated by establishing that T cells are positively selected to express a specificity for self major histocompatibility complex (MHC), and that those T cells bearing receptors potentially reactive to self antigen fragments, presumably presented by thymic MHC, are selected against. The precise mechanism by which tolerance is induced and the stage of T-cell development at which it occurs are not known. We have now studied T-cell tolerance in transgenic mice expressing a T-cell receptor with double specificities for lymphocytic choriomeningitis virus (LCMV)-H-2Db and for the mixed-lymphocyte stimulatory (MIsa) antigen. We report that alpha beta TCR transgenic mice tolerant to LCMV have drastically reduced numbers of CD4+CD8+ thymocytes and of peripheral T cells carrying the CD8 antigen. By contrast, tolerance to MIsa antigen in the same alpha beta TCR transgenic MIsa mice leads to deletion of only mature thymocytes and peripheral T cells and does not affect CD4+CD8+ thymocytes. Thus the same transgenic TCR-expressing T cells may be tolerized at different stages of their maturation and at different locations in the thymus depending on the antigen involved.     

Positive selection of CD4+ thymocytes controlled by MHC class II gene products

The mature T-cell antigen receptor repertoire is characterized by lack of reactivity to self-components as well as by preferential reactivity to foreign antigens in the context of polymorphic self-proteins encoded within the major histocompatibility complex. Whereas the former characteristic (referred to as negative selection or tolerance) is associated with intrathymic deletion of T cells expressing T-cell antigen receptor beta-chain variable (V beta) domains, which confer a preferential reactivity to self antigens, the existence of the latter (referred to as positive selection or MHC restriction) has so far only been inferred indirectly from functional studies. We show here that intrathymic deletion of V+beta 6 T cells (reactive with a self-antigen encoded by the Mlsa locus) is controlled by polymorphic MHC class II determinants. Furthermore, in mice lacking expression of Mlsa, the same class II MHC loci control the frequency of occurrence of V+beta 6 cells among mature CD4+ T lymphocytes. These data are direct evidence for positive selection by MHC determinants in the thymus in unmanipulated animals.     

Autoimmune diabetes as a consequence of locally produced interleukin-2.

During cell differentiation in the thymus, self-reactive T cells can be generated. The majority of these seem to be deleted after intrathymic encounter with the relevant autoantigen. As all self antigens are unlikely to be present in the thymus, some autoreactive T cells may escape censorship. Here we study the fate of these cells using transgenic mice expressing the class I molecule H-2Kb (Kb) in the insulin-producing beta-cells of the pancreas. These mice were crossed with mice transgenic for genes encoding a Kb-specific T-cell antigen receptor (TCR) which could be detected using a clonotype-specific monoclonal antibody. Although T cells expressing the highest level of transgenic TCR were deleted intrathymically in double-transgenic mice, Kb-specific T cells were detected in the periphery. These cells caused the rejection of Kb-expressing skin grafts, but ignored islet Kb antigens even after priming. But when double-transgenic mice were crossed with transgenic mice expressing the lymphokine interleukin-2 in the pancreatic beta-cells, there was a rapid onset of diabetes. These results indicate that autoreactive T cells that ignore self antigens may cause autoimmune diabetes when provided with exogenous 'help' in the form of interleukin-2.     

Positive and negative selection of an antigen receptor on T cells in transgenic mice

The T-cell repertoire found in the periphery is thought to be shaped by two developmental events in the thymus that involve the antigen receptors of T lymphocytes. First, interactions between T cells and major histocompatibility complex (MHC) molecules select a T-cell repertoire skewed towards recognition of antigens in the context of self-MHC molecules. In addition, T cells that react strongly to self-MHC molecules are eliminated by a process called self-tolerance. We have recently described transgenic mice expressing the alpha beta T-cell receptor from the cytotoxic T lymphocyte 2C (ref. 11). The clone 2C was derived from a BALB.B (H-2b) anti-BALB/c (H-2d) mixed lymphocyte culture and is specific for the Ld class I MHC antigen. In transgenic H-2b mice, a large fraction of T cells in the periphery expressed the 2C T-cell receptor. These T cells were predominantly CD4-CD8+ and were able to specifically lyse target cells bearing Ld. We now report that in the periphery of transgenic mice expressing Ld, functional T cells bearing the 2C T-cell receptor were deleted. This elimination of autoreactive T cells appears to take place at or before the CD4+CD8+ stage in thymocyte development. In addition, we report that in H-2s mice, a non-autoreactive target haplotype, large numbers of CD8+ T cells bearing the 2C T-cell receptor were not found, providing strong evidence for the positive selection of the 2C T-cell receptor specificity by H-2b molecules.     

Role of self-peptides in positively selecting the T-cell repertoire

The fate of an immature thymocyte is determined by the specificity of its alpha beta T-cell receptor. Only cells expressing receptors that interact with sufficient affinity with major histocompatibility complex (MHC) molecules expressed on thymus epithelial cells are positively selected and go on to mature and seed the peripheral lymphoid organs. The H-2Kb class-I MHC molecule positively selects for the maturation of cytotoxic T lymphocytes that will respond in the periphery to H-2Kb cells presenting a foreign peptide. We have now analysed the ability of variant H-2Kb molecules to positively select T-cells that respond to H-2Kb with ovalbumin. Our results indicate that self-peptides, presented in the groove of the class-I molecule on thymus epithelial cells, are critically involved in positive selection of the T-cell repertoire. Furthermore, the ability of four different H-2Kb variants to select this response in the thymus correlates with their ability to present the ovalbumin peptide, indicating that a self-peptide mimic of the foreign peptide could be involved in positive selection.     

Responses to the H-2Kba mutant involve recognition of syngeneic Ia molecules

Conventional antigens appear to be recognized by T lymphocytes only when associated with major histocompatibility complex (MHC) antigens. Using antigen-specific proliferation as a model for helper T lymphocytes, it has been demonstrated that Ly1+T cells recognize antigen presented in association with syngeneic Ia molecules. In contrast to responses to conventional antigens, however, a large number of studies have suggested that the stimulation of alloreactive Ly1+T cells, and helper T cells specific for allogeneic cytotoxic T lymphocyte (CTL) responses, involve the direct recognition of Ia alloantigens. For the generation of optimal allogeneic CTL activity it has been proposed that Ly1+T cells recognize allo-Ia antigens directly and provide help to pre-CTLs that respond to allo-H-2K and/or D determinants. Thus, the B6.C.H-2bm1 mutant (bm1, formerly referred to as Hz1), which is believed to consist of a substitution of two amino acids in the H-2Kb antigen, has presented a paradox, for it can stimulate strong mixed lymphocyte culture (MLC), graft versus host and CTL responses by T cells of H-2b haplotype mice in the apparent absence of any alloantigenic differences in the I region. We now present evidence that the stimulation of proliferative and helper T cells by the mutant B6.C.H-2bm1 results from the H-2Kba antigen being recognized in the context of syngeneic Ia determinants. Thus responses to both conventional antigens and allogeneic MHC gene products may proceed via the recognition of antigen in the context of self Ia molecules.     

Development of CD4-CD8+ cytotoxic T cells requires interactions with class I MHC determinants

Differentiation of bone marrow derived precursors into mature T cells takes place in the thymus. During differentiation, T cells develop the receptor repertoire which allows them to recognize antigen in the context of self major histocompatibility complex (MHC) molecules. Mature T helper cells (mostly CD4+ CD8-) recognize antigen in the context of class II MHC molecules, whereas cytotoxic T cells (mostly CD4-CD8+) recognize antigen in the context of class I MHC determinants. Thymic MHC-encoded determinants greatly influence the selection of the T-cell receptor repertoire. In addition to positive selection, a negative selection to eliminate self-reactive T-cell clones is thought to occur in the thymus, but how this 'education' occurs is not well understood. It has been suggested that during differentiation an interaction between the T-cell receptor (TCR) and MHC-encoded determinants occurs, leading to the selection of an MHC-restricted receptor repertoire. In support of this hypothesis, class-II-specific, CD4+ CD8- helper T cells fail to develop in mice neonatally treated with anti-class II monoclonal antibody (mAb). As CD4-CD8+ cells differ from the CD4+ CD8- lineage (in function, MHC-restriction specificity and perhaps site of education) we examined whether interactions with MHC determinants are also necessary for the development of class-I-specific T cells. Here we show that mice chronically treated with anti-class I mAb from birth lack CD4-CD8+ cells and cytotoxic T-cell precursors, indicating that most CD4-CD8+ T cells need interaction with class I MHC molecules during differentiation.     

Evidence from in vitro studies that tolerance to self antigens is MHC-restricted

Mature T cells respond to foreign antigens in the context of self major histocompatibility complex (MHC)-encoded products: T helper cells recognize antigen in the context of class II molecules, while cytotoxic T cells (CTL) recognize antigen plus class I molecules. Recent evidence suggests that the MHC-restricted T cell is unable to recognize either the foreign antigen or the self-MHC product alone, but only a complex of the two. Unresponsiveness to self antigens--self tolerance--implies the deletion or suppression of clones of T cells having reactivity to self antigens. Here we demonstrate the presence in normal mice of T cells which recognize self antigens together with allogeneic MHC products. This finding suggests the MHC restriction of T-cell recognition during the entire process of T-cell ontogeny, that is, MHC restriction of self tolerance.     

Developmental regulation of T-cell receptor gene expression

In contrast to B cells or their antibody products, T lymphocytes have a dual specificity, for both the eliciting foreign antigen and for polymorphic determinants on cell surface glycoproteins encoded in the major histocompatibility complex (MHC restriction). The recent identification of T-cell receptor glycoproteins as well as the genes encoding T-cell receptor subunits will help to elucidate whether MHC proteins and foreign antigens are recognized by two T-cell receptors or by a single receptor. An important feature of MHC restriction is that it appears to be largely acquired by a differentiating T-cell population under the influence of MHC antigens expressed in the thymus, suggesting that precursor T cells are selected on the basis of their reactivity with MHC determinants expressed in the host thymus. To understand this process of 'thymus education', knowledge of the developmental regulation of T-cell receptor gene expression is necessary. Here we report that whereas messenger RNAs encoding the beta-and gamma-subunits are relatively abundant in immature thymocytes, alpha mRNA levels are very low. Interestingly, whereas alpha mRNA levels increase during further development and beta mRNA levels stay roughly constant, gamma mRNA falls to very low levels in mature T cells, suggesting a role for the gamma gene in T-cell differentiation.     

Positive selection of CD4-CD8+ T cells in the thymus of normal mice

The diversification of the repertoire of T-cell antigen receptor (TCR) specificities is influenced by at least two selection processes which occur in the thymus. One of these, termed 'negative selection', is required to install a state of tolerance to self-antigens in the T-cell repertoire and is often achieved by clonal deletion. The second type of selection operating in the thymus results in preferential differentiation of T cells that have restriction specificity for thymic major histocompatibility complex glycoproteins, but the mechanisms leading to this selective process are not yet clear. One model used to describe this 'positive selection' proposes that only those T cells with sufficient avidity for the MHC glycoproteins expressed in the thymus are allowed to acquire functional competence. Here we directly investigate the generation of TCR specificities by following the fate of developing V beta 17+ CD4-CD8+ T cells under conditions where one of the main class I-MHC molecules, either H-2K or H-2D, was specifically blocked by in vitro monoclonal antibody treatment. The results show that development of V beta 17+ CD4-CD8+ T cells in the SJL H-2s mouse strain is selectively abrogated by blocking class I-Ks molecules but is unaffected by blocking class I-Ds molecules. These data directly demonstrate that generation of CD4-CD8+ T cells expressing a particular TCR V beta segment can be correlated with the expression of a particular class I-MHC molecule, thereby providing evidence for positive selection.     

Minor but not major histocompatibility antigens of thymus epithelium tolerize precursors of cytolytic T cells

Treatment of fetal thymuses with 2-deoxyguanosine depletes these organs of many haematopoietic cells, and if such thymuses are transplanted into allogeneic athymic nude mice, intrathymic development of cytolytic T-lymphocyte precursors (CTL-P) occurs, including those which are specific for class I major histocompatibility complex (MHC) antigens expressed by the thymus epithelium. Thus, T cells from BALB/c (H-2d) nude mice transplanted with allogeneic C57BL/6 (H-2b) thymic epithelium can be stimulated in vitro to produce CTL specific for H-2b class I MHC antigens. We report here that thymocytes and lymph node T cells from such mice are responsive in mixed leukocyte reaction in the absence of exogenous growth factors, indicating that lack of tolerance is manifest at the level of CTL-P and proliferating T cells. We also show that T cells from such mice are tolerant to minor histocompatibility antigens of the thymus donor in the context of MHC antigens of the recipient. The results indicate that haematopoietic rather than epithelial cells tolerize CTL-P and that donor-type minor but not major histocompatability antigens can be presented in tolerogenic form by haematopoietic cells expressing recipient-type MHC antigens.     

Expression and function of interleukin-2 receptors on immature thymocytes

T-cell differentiation represents a unique system for studying mechanisms of lymphoid development because it occurs in a segregated site, the thymus, in which distinct subpopulations of thymocytes at various stages of differentiation can be defined on the basis of the differential expression of T-cell surface antigens as well as topography. There is particular interest in thymocyte differentiation because the genotype of radioresistant thymus cells influences the specificity repertoire of the pool of T cells that mature therein: that is, the major histocompatibility complex (MHC) antigens expressed by thymus cells bias the pool of maturing T cells towards recognition of antigens in the 'context' of the products of that MHC haplotype ('thymus education'; refs 1-3). Immature T cells with affinity for thymus MHC antigens are generally thought to undergo a stage of positive selection in the thymus. Here we report that 30% of cells in the least mature adult thymocyte subpopulation yet defined, as well as 50% of immature fetal thymocytes, express receptors for interleukin-2 (IL-2, the T-cell growth factor) without in vitro induction, and will proliferate vigorously in an IL-2-dependent fashion if provided with co-stimulating mitogen.     

Tolerance of class I histocompatibility antigens expressed extrathymically

Although convincing evidence has been obtained for the imposition of self-tolerance by the intrathymic deletion of self-reactive T cells, the development of tolerance to antigens which are expressed only in the periphery is not so well understood. We have approached this question by creating transgenic mice which carry a class I major histocompatibility complex (MHC) gene (H-2Kb) linked to the rat insulin promoter. Mice expressing the transgene develop diabetes, but do not appear to mount an immune response against the transgene-expressing pancreatic beta-cells, even when the transgene is allogeneic with respect to the endogenous host H-2 antigens. We have now explored the mechanism of this tolerance further. We find that spleen cells from pre-diabetic transgenic (RIP-Kb) mice do not kill targets bearing H-2Kb, whereas thymus cells from the same mice do. The unresponsiveness of these spleen cells can be reversed in vitro by providing recombinant interleukin-2 (rIL-2). In older, diabetic mice, responsiveness develops as the pancreatic beta-cells are lost. Our results point to an extrathymic mechanism of tolerance induction, dependent on the continuous presence of antigen and the lack of IL-2 in the local environment of potentially reactive T cells.     

T-cell tolerance by clonal anergy in transgenic mice with nonlymphoid expression of MHC class II I-E

T-cell reactivity to the class II major histocompatibility complex I-E antigen is associated with T-cell antigen receptors containing the V beta gene segments V beta 17a and V beta 5. Mice expressing I-E with the normal tissue distribution (on B cells, macrophages, dendritic cells and thymic epithelium) induce tolerance to self I-E by clonal deletion in the thymus. By contrast, we find that transgenic INS-I-E mice that express I-E on pancreatic beta-cells, but not in the thymus or peripheral lymphoid organs, are tolerant to I-E but have not deleted V beta 5- and V beta 17a-bearing T cells. Moreover, whereas T-cell populations from nontransgenic mice proliferate in response to receptor crosslinking with V beta 5- and V beta 17a-specific antibodies, T cells from INS-I-E mice do not. Thus, our experiments provide direct evidence that T-cell tolerance by clonal paralysis does occur during normal T-cell development in vivo.